Liquid handling pipettors are an essential tool and are used extensively in laboratories across but not limited to such industries as academic research, applied testing, and medical diagnostics.
The purpose of the pipettor is to transfer specific volumes of liquid between containers. Examples of this action include but are not limited to: the sub-sampling of patient liquid samples such as blood or urine from a patient sampling vessel to an analysis vessel such as the 96-well microtiter plate; the assembly of individual reagents from master stocks to a tube containing a mixture or reagents such as would be used for polymerase chain reaction (PCR); and transfer of tissue culture growth media from stock to individual cultures.
Pipettors by design are accurate at measuring specific volumes of liquids. The volumes of liquids measured can range from nanoliters to milliliters. Because of the extensive range of volumes measured, pipettors are manufactured to cover a subset of volumes such as but not limited to: less than 1 microliter, 1-2 microliters, 1 to 10 microliters, 10 to 100 microliters, 20 to 200 microliters, 100 to 1000 microliters, 1 milliliter to 5 milliters.
In order for pipettors to accurately measure specific volumes, they must be routinely tested and calibrated. This routine testing and calibration is extremely important and often specified in a laboratory's Standard Operating Procedure (SOP). Evidence of routine verification and calibration of pipettors may even be subjected to audit by third party organizations such as those that regulate medical diagnostics and applied testing laboratories.
Verification and calibration of pipettors is most usually done gravimetrically, by weighing the dispensed amount of a reference liquid such as water. This method requires the use of an extremely accurate set of weighing scales, such as a 6 decimal point scale used to measure weights as small as 1 microgram. These scales are themselves verified and calibrated against an external weight reference or calibration device that has been certified as true and accurate measure of weight.
Another commonly used method is an absorbance-based system that utilizes a photometer and dual color dye as the basis of pipettor calibration verification. One such system uses a known concentration of a dye that absorbs at one wavelength. The photometer determines the vial pathlength, and then a dye solution of known concentration and a different absorbance maximum is pipetted (using the pipettor to be calibrated) into the reference solution. The solutions are mixed by the photometer and the absorbance is read. The photometer and software convert the absorbance reading to the volume pipetted, and the result is printed. After the user has taken the desired number of data points with the pipettor, the device generates a printed result with statistical data that comprises individual sample volumes or replicates, mean volume of all replicates, % CV (precision), and inaccuracy from target volume. The machines necessary for this method are fairly expensive.
The calibration process is arduous, time consuming, and prone to inaccuracies, particularly at small volumes where evaporation of water and the propensity for water to adhere to plastics can introduce error. At these lower volumes, the movement of air currents across the pan of a sensitive scale, or slight vibrations transmitted through a building's structure, can also introduce error in gravimetric analyses.
A typical gravimetric process involves repetitive weighing of a series of water volumes appropriate to the dispensation range of the pipettor, e.g. 2 microliters, 10 microliters and 20 microliters for a pipettor with a stated range of 2-20 microliters. These measurements are often in the range where environmental inaccuracies described above are significant (generally <100 microliters). The process would be repeated at least 6 times to obtain a reliable accuracy for each volume: for example, 100 microliters±2.0 microliters. At this volume the pipettor would be said to have an accuracy of 100 microliters with a critical variance (CV) of ≤2%.
The pipettor is fitted with a disposable plastic tip that contacts the liquid being transferred. The underlying mechanism of the pipettor is a piston housed in a cylinder. As the piston moves downwards in the cylinder air is displaced. The piston is depressed to a specified distance by the action of the thumb pushing downwards on the top of the piston or by a small electric motor and a drive assembly such as a worm gear. The pipette tip is inserted into the liquid to be transferred and then the piston allowed to return to its original position; the displaced air is replaced by liquid, filling the pipette tip with the required volume of liquid. The pipette and tip are moved to the target vessel and the piston again depressed and the liquid ejected. Over-pipetting, i.e. depressing the piston slightly further than before the liquid was aspirated, ensures that all liquid within the pipette tip is ejected. The piston is returned to its pre-pipetting position and the disposable pipette tip ejected.
The starting position of the piston and the distance the piston travels is set by the user before the liquid is aspirated. The start position of the piston is set via a volume control wheel for manual setting or digitally for a motor-driven electronic pipettor. The user simply adjusts the volume control wheel or the digital display to display the intended volume to be transferred. The user proceeds to transfer the nominated liquid volume as described above.
Current verification and volume calibration involves confirming, by gravimetric or dye absorbance methods, that the selected volume shown on the pipettor and the volume of liquid aspirated and dispensed are identical with respect to target volume and CV of variance. If there is disagreement between the selected volume to be dispensed and the actual volume dispensed, then the pipettor is out of calibration. Before the pipettor can be used for routine laboratory use it must be calibrated to insure the accuracy of the volume dispensed with respect to the selected volume shown on the pipettor.
While others have developed methods for collecting selected volumes of liquids using the geometry of the capillary-type device, such as Kenney in U.S. Pat. No. 6,531,098 and Karg et al. in U.S. Pat. No. 8,080,218, no one has invented an easy method of determining whether a desired volume of liquid is accurately dispensed.
The invention described eliminates the arduousness of determining if a pipettor is within acceptable accuracy limits. It entails a simple method of validating the calibration of the pipettor, and, within the accuracy limits of the device, performing the calibration. In addition, while current methods of calibrating pipettors indirectly determine the volume dispensed, either by determining weight or the absorbance of a dye contained in the dispensed liquid, this invention directly measures the volume dispensed by the pipettor. Thirdly, this invention allows for high throughput automation, with the device taking the form of a standardized microwell or microtiter plate.